**3. Bioethanol production**

Bioethanol is an alcohol made by fermenting the rich sugar components of biomass which is seen as a good fuel alternative. The use of bioethanol as a biofuel has very important advantage – it is generally CO2 neutral. This is achieved because in the growing phase of the biomass plants, CO2 is absorbed and then released in the same volume during combustion of the fuel (Stephenson et al., 2010). This creates an obvious advantage over fossil fuels which only emit CO2 as well as other poisonous gasses. Bioethanol can be used as a fuel for transport in its pure form, but it is usually used as a gasoline additive to increase its octane rating and improve vehicle efficiency (Balat & Balat, 2009).

Nowadays, the bioethanol market has continued to grow rapidly, for example, from about 46 billion L of ethanol produced worldwide in 2007 to the expected value of 100 billion L in 2015 (Balat & Balat, 2009; Sarkar et al., 2012). The USA is the world leader in the production of bioethanol with 48 billion L in 2009 (Muthaiyan & Ricke, 2010), followed by Brazil with 27,0 billion L in 2009 (Soccol et al., 2010) which determined 62% of the worldwide

play an important role in granulation phenomena and was found to be a component of essential enzymes that carry out numerous anaerobic reactions (Vlyssides et al., 2009; Yu et al., 2000). The conversion of COD to biogas components and bacterial growth may be limited at iron deficient concentrations. However, the accumulation of iron ions may decrease the specific activity of the bacterial groups, including methanogens (Yu et al., 2000). It was reported that high Fe2+ concentration in the anaerobic sludge granules led to decrease of the specific activity of biomass due to the presence of a large amount of minerals deposited within the granules, a significant decrease in the water content in granules, and the possible toxicity of high-concentration Fe2+ accumulated inside the granules (Yu et al., 2000). During the experiment, biogas production rate was not decreased from Stage 1 to 4, which could indicate that the activity of methanogenic bacteria was not inhibited by anaerobic steel corrosion process. The maximum value for biogas rate was 8.22 L d-1 in RFe and 4.2 L d-1 in R0. Najafpour et al. (2008) achieved the biogas production of 3.6 L d-1 for HRT of 48 h with the methane content of 76% from UF whey permeate. Venetsaneas et al. (2009) achieved about 1 L CH4 d-1 and 68% v/v methane content in biogas in the two-stage

On the basis of this study, it is expected that the UASB reactors packed with steel elements may be applicable to treat UF whey permeate to produce biogas with high CH4 content. The COD removal efficiency, biogas productivity and CH4 content in biogas were enhanced by 11.4 - 17.0% (p<0.05), 1.67 - 2.15 m3 m3 d-1 (p<0.05), 8.3 - 17.2% (p<0.05), respectively, in the UASB reactor packed with steel elements compared to the control reactor performances. In this work, the maximum biogas production rate was 8.22 L d-1 in the reactor containing additional iron medium in contrast to about 4.2 L d-1 in the control reactor. Total phosphorus removal efficiency obtained in RFe was higher by 58.4 - 77.7% than in R0 (p<0.05). High iron concentration in the anaerobic granular sludge was not contributed to inhibit the activity of methanogenic bacteria. It should be pointed that during anaerobic corrosion process a protective layer on the steel surface can be formated to decrease

Bioethanol is an alcohol made by fermenting the rich sugar components of biomass which is seen as a good fuel alternative. The use of bioethanol as a biofuel has very important advantage – it is generally CO2 neutral. This is achieved because in the growing phase of the biomass plants, CO2 is absorbed and then released in the same volume during combustion of the fuel (Stephenson et al., 2010). This creates an obvious advantage over fossil fuels which only emit CO2 as well as other poisonous gasses. Bioethanol can be used as a fuel for transport in its pure form, but it is usually used as a gasoline additive to increase its octane

Nowadays, the bioethanol market has continued to grow rapidly, for example, from about 46 billion L of ethanol produced worldwide in 2007 to the expected value of 100 billion L in 2015 (Balat & Balat, 2009; Sarkar et al., 2012). The USA is the world leader in the production of bioethanol with 48 billion L in 2009 (Muthaiyan & Ricke, 2010), followed by Brazil with 27,0 billion L in 2009 (Soccol et al., 2010) which determined 62% of the worldwide

process for cheese whey fermentation.

phosphorus removal efficiency.

**3. Bioethanol production** 

rating and improve vehicle efficiency (Balat & Balat, 2009).

**2.3 Conclusions** 

production (Sarkar et al., 2012). In the USA, bioethanol is mainly used as a 10% petrol additive (E10 is the standard petrol fuel, in 2011 introduced E15). In Brazil, it is offered both as a pure fuel (E100) and is blended with conventional petrol with a content of 20 to 25% (E20, E25). In Europe, with the adoption of the Biofuel Directive 2003/30/EC in 2003, the framework conditions were especially created for European bioethanol production. Today France is a leading producer of bioethanol, then Germany, Spain, Sweden and Dutch are the significant producers in Europe (Gnansounou, 2010). Current large scale production of fuel ethanol is mainly based on sugarcane (Brasil), corn (the USA), sugar beet and wheat (Europe), (Balat & Balat, 2009). The recent rise in the prices of food ethanol biomass has shifted in focus towards a possibility of deriving fuel ethanol from any type of biomass, especially cellulosic biomass (corn or wheat straw, sugarcane bagasse, wood, grass) and food waste biomass (organic waste and wastewater from food processing industries) (Sarkar et al., 2012; Soccol et al., 2010).

According to the literature, cheese whey could be a suitable substrate for bioethanol production (Kourkoutas et al., 2002; Zafar & Owais, 2006). Lewandowska & Kujawski (2007) used a solution of dried UF whey permeate as a substrate for semi-continuous ethanol fermentation. Silveira et al. (2005) fermented the solution of UF whey permeate in batch cultures. Ghaly & El-Taweel (1997) developed a kinetic model for continuous ethanol fermentation from lactose. Moreover, in 2008 there were two industrial scale whey-ethanol plants in the United States which produced 8 million gallons of fuel ethanol per year (Ling, 2008). In New Zealand there were whey-ethanol plants with an annual production of about 5 million gallons of ethanol (Ling, 2008). Industrial-scale plants producing bioethanol form whey permeate are operated in Ireland (de Glutz, 2009).

There are many reports of potential applications of yeast strains in ethanol production from UF whey permeate streams, but most of them focused on *Kluyveromyces sp.* due to its ability to directly ferment lactose (Kourkoutas et al., 2005; Ozmhc & Kargi, 2008; Silveira et al., 2005; ). These yeasts generally suffer from low conversion yields (0.4 kg ethanol kg-1 lactose) and are very sensitive to product (ethanol) inhibition at concentrations as low as 20 g L-1 (de Glutz, 2009). An alternative is to employ indirect fermentation yeasts, such as *Saccharomyces cerevisiae*, which show considerably better ethanol fermentation performance (0.520 kg ethanol kg-1 lactose) and much higher alcohol tolerance (100 - 120 g L-1) (Coté et al., 2004; de Glutz, 2009). The disadvantage of using *S. cerevisiae* is the inability to directly ferment lactose. It can be solved by genetic manipulation of yeasts or facilitate the process with a simultaneous lactose hydrolysis, for example by co-immobilization of yeast cells with the enzyme (Coté et al., 2004; Guimarães et al., 2008). Moreover, higher ethanol production could be achieved by application of different stimulation processes, improving biological activity of yeasts. Many researchers have found that ultrasonic stimulation has the function of promoting the activity of enzyme, cell growth and cell membrane permeability (Chisti, 2003; Liu et al., 2003a; Liu et al., 2007; Schläfer et al., 2000). However, application of ultrasonic irradiation at improper intensity or period has destructive impact on cells by disrupting the cell membranes and deactivating biological molecules such as enzymes or DNA (Liu et al., 2007).

The objectives of the studies were: (1) to investigate bioethanol production from UF whey permeate in continuous fermentation in UASB reactors by *K. marxianus* 499, (2) to evaluate the effects of low intensity ultrasound (20 kHz, 1 W L-1) for ethanol production from UF whey permeate by *S. cerevisiae* B4.

Feasibility of Bioenergy Production from

**3.1.2 Results and discussion** 

whey within 22 h by *K. marxianus*.

Lactose consumption (%)

consumption

Kujawski, 2007).

Ultrafiltration Whey Permeate Using the UASB Reactors 201

The effects of HRTs on the lactose concentration in the effluent distillate and percent lactose consumption are shown in Fig. 5. When the HRT was 12 h, the average lactose concentration in the effluent distillate was as high as 25 g L-1 and the average lactose utilization efficiency was only 50%. Increasing the HRT from 12 to 24 h increased the average yield of lactose utilization to 85%. Further increase in the HRT from 24 to 48 h resulted in the highest lactose utilization of 95%. Similar results obtained Ghaly & El-Taweel (1997). They observed 98% lactose utilization for continuous fermentation from cheese whey with 50 g L-1 initial lactose concentration at the HRT of 42 h using the yeast strain of *Candida pseudotropicalis*. Kargi & Ozmihci (2006) reported complete fermentation of lactose (35 g L-1 initial lactose concentration) in cheese whey powder (CWP) solution using the yeast strain of *K. marxianus* at HRT of 48 h. Zafar & Owais (2006) obtained about 86% lactose utilization from crude

12 24 48

Lactose consumption Lactose remaining

HRT (h)

According to Ghaly & El-Taweel (1997) lower lactose fermentation efficiency under low HRT could be attributed to the cell washout phenomenon and the low cell numbers in the reactor chamber. To remove this problem, during this experiment, the reactors were provided with G-L-S separator and the immobilization of yeast culture was done. The immobilization process made ethanol production more efficient compared to the free system and prolonged the activity of yeast cells (Kourkoutas et al. 2004), which is especially important in continuous fermentation processes. Moreover, the application of immobilization process reduced the risk of microbial cells infection when the yeasts were cultivated on the fermentation medium that was not sterilized before use (Lewandowska &

Fig. 5. Effects of HRT on the lactose concentration in the effluent and percent lactose

0

5

10

15

Lactose remaining (g L-1)

20

25

30
